Rapid method for separation of small molecules using reverse phase high performance liquid chromatography

ABSTRACT

Disclosed is a rapid method for the separation of small organic compounds using gradient reverse phase HPLC. The method achieves a run time of one minute or less and a resolution with a peak production of at least 1 peak/2 seconds. The method is also able to achieve the separation of a compound from a mixture of compounds in an elution sample having a volume of 2 milliliters of less.

REFERENCE TO RELATED APPLICATIONS

This application is a 371 of PCT/US99/02371 filed Feb. 3, 1999, which,in turn, claims the benefit of U.S. application Ser. No. 09/028,401,filed Feb. 24, 1998, now U.S. Pat. No. 5,968,361, and claims the benefitof U.S. Provisional Application No. 60/073,489, filed Feb. 3, 1998.

FIELD OF THE INVENTION

The invention relates to the field of high performance liquidchromatography and more particularly to the field of high performanceliquid chromatography separation of small organic molecules.

BACKGROUND OF THE INVENTION

High-performance liquid chromatography (HPLC) is commonly used foranalytical and preparative separations of biopolymers and other organicmolecules. For instance, the individual components within a complexorganic reaction mixture may be separated by HPLC. HPLC is performed ina pressure-resistant tube containing a stationary adsorbent which is thepacking material. A pressure mechanism exerts pressure on a mobile phaseapplied to one end of the column and moves it through the column causingit to exit the opposite end of the column. A sample containing a mixtureof compounds is injected onto the column through a sample injectionport. As the sample moves through the packing material, the variouscomponents of the sample adsorb to the packing material with differentaffinities. The components, therefore, can elute from the columnseparately under appropriate conditions. On a reverse phase HPLC columnthe compounds within a sample are separated based on hydrophobicity.

HPLC analysis may be performed in isocratic or gradient mode. Anisocratic HPLC separation is one which is carried out under a constanteluant composition. A gradient HPLC separation is characterized by agradual change in the percentage of two or more solvents applied to thecolumn over time. The change in solvent often is controlled by a mixingdevice which mixes solvent A and solvent B to produce the HPLC solventjust prior to its movement through the column. The amount of time overwhich the gradient is changed from one extreme to the opposite extremeis the gradient time.

Generally in gradient chromatography it is believed that increasing theflow rate and/or decreasing the gradient time results in a loss ofresolution, that is the ability of the column to separate the componentswithin the mixture into discrete eluant fractions. (Snyder, L., et al.,“Practical HPLC Method Development”, Wiley-Interscience Publication,(1997).

Rapid methods for the preparation and isolation of potential drugcandidates using automated synthetic organic chemistry techniques tocreate combinatorial libraries represents an important advance in drugdiscovery. Certain combinatorial libraries encompass a series ofcompounds having common structural features but which differ in thenumber or type of group attached to the main structure. Each compoundwithin a combinatorial library created by parallel synthesis is aseparate sample housed in a tube or well of a microtitre plate. Once thelibrary is completed, each sample is subjected to quality controlanalysis to confirm that the particular sample includes the desiredlibrary component at the requisite purity. Generally this isaccomplished by subjecting the samples to HPLC with UV, evaporativelight scatter detection (ESLD), or mass spectrometry detection; IR; NMR;or any other appropriate analytical techniques. The qualitative analysisof such combinatorial libraries by conventional HPLC requires on theaverage 5 to 20 minutes in order to separate various compounds withinthe sample.

A problem encountered with prior art methods for separation of compoundsin combinatorial libraries using HPLC is the length of time required forseparation of each sample. Each sample of a combinatorial libraryproduced by parallel synthesis must be analyzed separately to determineif that sample houses the appropriate compound and/or to separate thecompounds in the mixture. Each library includes thousands of sampleseach of which require an average run time of 10 minutes. The amount oftime required to perform separations on these samples may run on theorder of months using standard equipment and methodology.

SUMMARY OF THE INVENTION

The present invention provides rapid methods for the analysis andpreparative isolation of relatively simple synthetic mixtures containinga small number of reagents, the product(s) of interest and a relativelysmall number of side products using HPLC. The methods of the inventionreduce the HPLC analysis run time per sample from an average of 5-20minutes shown in the prior art (Weller, et al., Molecular Diversity,(1997), 3:61-70) to less than one minute without a meaningful loss ofresolution. The invention depends in part upon the discovery that smallorganic molecules could be separated on a full gradient reverse phaseHPLC by minimizing the total volume of eluant applied to the column,maximizing the linear flow velocity of the eluant and compressing thegradient time to resolve a peak at least every 2 seconds. A fullgradient is defined as a change in the solvent B concentration of atleast 50%. For example, if the initial concentration of solvent B was15%, a full gradient would be achieved when the concentration of solventB reached 65%. The prior art believed that if the total eluant volumewas decreased and the flow rate increased to the levels indicated in theinvention, the resolution of the peaks eluting off the column would besignificantly decreased to an extent that it would not be possible toobtain a discrete separation of a mixture of small organic compounds.

The methods of the invention include applying a mixture of compounds toa reverse phase column configured in a gradient high performance liquidchromatography system, and operating with a flow rate of at least 5column volumes/min. A complete gradient is applied to the column at arate which uses a maximum total volume of 10 column volumes; preferably5 column volumes in order to maximize speed. These parameters allow eachsmall organic component within the mixture of compounds to elute in adistinct fraction from the column with sufficient resolution whichpermits a peak production of at least 1 peak/2 seconds. A one minuteanalysis, using a peak production of 1 peak/2 seconds, would translateinto an analysis which could baseline separate more than 30 individualpeaks.

The amount of time that the complete separation requires depends on theparameters used in the separation, such as the length of the column andthe amount of solvent used. Preferably the mixture of compounds isapplied to the column at a first time point and all the compounds areeluted within a time period of less than one minute from the first timepoint. In other preferred embodiments all compounds are eluted within atime period of less than 30 seconds. In other embodiments all compoundsare eluted within a time period of less than 20 seconds.

In one embodiment of the invention the method also includes the step ofdetecting at least one of the compounds as it elutes from the column. Inanother embodiment the method includes the step of collecting at leastone of the compounds in a distinct fraction as it elutes from thecolumn.

In preferred embodiments, the mixture of molecules includes reactantsand a substantially pure product of the reactants.

In other preferred embodiments, including those listed above, the columnis less than or equal to 30 mm in length. The column is less than orequal to 15 mm in length in other embodiments.

According to other preferred embodiments, including those listed above,the column has a packing material which has an average diameter of lessthan 5 microns.

In other preferred embodiments, including those listed above, the peakproduction is at least 1 peak/1 second. The peak production is at least1 peak/0.5 seconds in other embodiments.

Preferably the total volume of liquid applied to the column per analysisis less than 15× column volumes, preferably less than 8× column volumes.The total volume of liquid may include a cleaning volume having amaximum of 2× column volume. In a preferred embodiment the total volumeof liquid may include an equilibration volume having a maximum of 1×column volume.

In other embodiments, including those listed above, the mixture ofmolecules is a member of a combinatorial library of small organicmolecules. Preferably the combinatorial library is made by means ofparallel synthesis methods and the method is performed for highthroughput purification and/or quality control analysis.

In additional embodiments the column flow rate has a linear velocity ofat least 3 mm/sec. In another embodiment the linear velocity is 5mm/sec.

The small organic compounds which elute from the column are typicallyanalyzed by a detection device such as a UV detector. In one embodimentof the invention the small organic molecules are analyzed by both a UVdetector and a mass spectrometer.

In one embodiment the method is a method for analysis of at least onecompound in the mixture of compounds. Preferably less than 10 μg of themixture of compounds is applied to the column for the analysis. In anembodiment the sample of compounds is not collected for further useafter it is eluted from the column.

In another embodiment the method is a method for preparative isolationof at least one compound in the mixture of compounds. Preferably between1 and 100 mg of the mixture of compounds is applied to the column forthe analysis. In an embodiment the compounds are collected in separatefractions for further use after they are eluted from the column.Preferably the at least one compound is collected in a fraction having avolume of 2 milliliters or less.

The high performance liquid chromatography may be performed at atemperature of greater than 20° C. in one embodiment. In anotherembodiment the method is performed at a temperature of greater than 50°C. In a preferred embodiment the method is performed at a temperature ofgreater than 60° C.

In another aspect the invention is a rapid high performance liquidchromatography method for the preparative isolation of a concentratedfraction of a small organic compound from a mixture of compounds. Themethod includes the steps of applying the mixture of compounds to areverse phase column in a gradient high performance liquidchromatography system, wherein the column has a flow rate of at least 5column volumes/min, applying a complete gradient to the column in amaximum volume of 10× column volume, causing the small organic compoundto elute in a distinct fraction, separate from the other molecules, fromthe column such that the elution permits resolution with a peakproduction of at least 1 peak/4 seconds and collecting the small organiccompound. The small organic compound, in some embodiments is collectedin a fraction having a maximum volume of 2 milliliters.

In other embodiments, including those listed above, the mixture ofmolecules is a member of a combinatorial library of small organicmolecules. Preferably the combinatorial library is made by means ofparallel synthesis methods and the method is performed for highthroughput purification and/or quality control analysis.

Each of the limitations of the invention can encompass variousembodiments of the invention. It is, therefore, anticipated that each ofthe limitations of the invention involving any one element orcombinations of elements can be included in each method.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic representation of the instrumentation to performthe method of the present invention.

FIG. 2 is a graph depicting the composition of the mobile phase duringthe HPLC analysis in terms of the number of column volumes of eluantapplied to the column.

FIG. 3 is a chromatogram of a test mixture obtained using the method ofthe present invention with less than 1 minute total analysis time.

FIG. 4 is a chromatogram of a test mixture obtained using the method ofthe present invention with less than ½ minute total analysis time.

FIG. 5 is a comparison of the chromatograms obtained for a samplesynthesized by parallel synthesis using a 20 minute, a 1 minute, and a30 second analysis.

FIG. 6 is another example of a comparison of the chromatograms obtainedfor a sample synthesized by parallel synthesis using a 20 minute, a 1minute, and a 30 second analysis.

FIG. 7 is a series of chromatograms obtained using the method of thepresent invention in order to determine peak capacity obtained withvarious flow rates using a 50×4.6 m, 3 μm Prontosil C18-SH column: (7A)flow rate of 2 ml/min; (7B) flow rate of 3 ml/min; and (7C) flow rate of4 ml/min.

FIG. 8 is a chromatogram obtained from a preparative isolation of 10 mgof each of the three standard compounds using a 20 mm×50 mm columnpacked with 5 um particles and a flow rate of 80 ml/min.

FIG. 9 is a chromatogram obtained from a preparative isolation ofapproximately 5 mg of a mixture prepared by parallel synthesis using theconditions identified in FIG. 8.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides new methods for the separation of smallorganic molecules using reverse phase HPLC with applications for theanalysis and/ore preparative isolation of the separated compounds.Although the underlying principles of analytical and preparative HPLCare the same (i.e., separating mixtures into discrete components),traditionally the mode of operation has been different. The goal ofanalytical HPLC has been focused on obtaining optimum resolutionutilizing a minimum amount of material, whereas preparativechromatography has been focused on loading the maximum quantity ofmaterial that could satisfactorily be resolved. Moreover, sinceisolation of a particular component is of interest in preparativechromatography, the eluant is collected after separation. This dualityof operation was reasonable when the analysis or preparative isolationwas for a small number of compounds, where each analysis could becustomized. However, using this same approach to analyze and/or purify alarge number of different compounds synthesized by parallel synthesis istoo costly and time consuming. In order to maintain a high throughputoperation for both analytical and preparative isolation, new fastermethods of the invention have been developed where the differencebetween analytical and preparative approaches are only in the scale ofthe equipment. By appropriately scaling up the column dimensions and theflow rate, the same separation, having the same resolution, can beachieved by loading 1 to 100 mg on a preparative column as can beachieved with 1 to 10 ug loading on an analytical column.

As used herein a “method for analysis” of small organic compounds is amethod in which a sample of small organic compounds is loaded on acolumn and performed according to the methods of the invention describedherein. In the method for analysis the sample may or may not becollected. In a preferred embodiment when the analysis method isperformed the sample is not collected. Preferably the method foranalysis involves the step of loading less than 20 μg, and even morepreferably less than 10 μg, of sample on the column.

As used herein a “method for preparative isolation” of small organiccompounds is a method in which a sample of small organic compounds isloaded on a column and performed according to the methods of theinvention described herein and wherein the compounds are collected infractions as they elute from the column. Preferably the method forpreparative isolation involves the step of loading less than 100 mg, andeven more preferably between 1 and 100 mg, of sample on the column.

The new methods of the invention include both analytical and preparativeseparations and are significantly faster than prior art methods. Themethods of the invention are particularly advantageous for separatingcomponents in small molecular weight combinatorial libraries as part ofthe quality control analysis and/or purification often conducted forsuch libraries. Prior to the invention each sample of certaincombinatorial libraries required approximately 5-20 minutes forseparation by HPLC. (Weller, et al., Molecular Diversity, (1997),3:61-70) Using the method of the invention, it has been discovered,surprisingly, that the separation time per sample can be reduced to lessthan one minute. The time reduction significantly increases the numberof samples which can be separated per unit time per instrument. At aminimum, the invention reduces the time of separation by a factor offive over prior art methods, enabling the separation of at least fivetimes as many compounds. In preferred embodiments, the methods are morethan 10 times faster than the prior art methods. Using the prior artmethods which typically require a total HPLC run time of 10 minutes onfully automated equipment, approximately 2,000 samples can be separatedper month. Using the methods of the present invention, which onlyrequire a run time of 1 minute, 20,000 samples can be separated in thesame time period. The new methods are described in detail below.

FIG. 1 illustrates the instrumentation useful according to the generalmethod of the invention. Two solvent reservoirs, 12 and 14, housingsolvents A and B are pumped by pumps 10 and 10A through tubes 18 a and18 b, respectively, into mixing chamber 16. A computer 32 controls theamount of solvent A and B which is pumped into the mixing chamber overtime. Solvents A and B are mixed in the mixing chamber to form ahomogenous solvent which passes through tube 18 c to a high pressurevalve 19 and into a column 20 containing a reverse phase packingmaterial referred to as the stationary phase. A plurality of samples arehoused within a microtitre plate 24 which rests in an auto injector 22which is also connected to the switching valve by tubing 18 f. Thesample in each well of the plate 24 is transported via tubing 18 fthrough the switching valve and then to the column through tubing 18 dby automated means. Once the sample enters the column, the small organiccompounds within the sample adsorb to the stationary phase withdifferent affinities based on the hydrophobicity of the compound. Afterthe sample is loaded the proportion of solvent A and solvent B isshifted with respect to time in order to create a gradient of solventthat is passed over the column. At certain points within the gradient,different small organic molecules are eluted from the column and carriedto a detection device. The eluant is passed through a detection devicesuch as a UV detector 28 to characterize the compounds within theeluant. In some cases the eluant is exposed to multiple detectors suchas a UV detector and a mass spectrometer 30. If isolation of aparticular component is of interest, the eluant from the UV detector issplit into a minor portion that goes to the mass spectrometer via tube18 g and a major portion which is transferred to the fraction collector(31) through tube 18 i. The equipment necessary to practice the presentinvention can be assembled from commercially available devices. Althoughsome of these devices were actually designed for other functions relatedto HPLC analysis, they can easily be adapted to the functions describedherein. For instance the 50 μl mixing chamber which is shown in apreferred embodiment for mixing eluant in the analytical setup isactually a chamber which is ordinarily used for post columnderivitization purposes. Additionally, the other devices which areassembled to produce the equipment are used according to the preferredembodiment of the invention and are adjusted as described in more detailbelow to produce the equipment useful for practicing the method of theinvention.

The methods of the invention depend in part upon the discovery thatsmall organic molecules could be analyzed and purified on a completegradient reverse phase HPLC by minimizing the volume of liquid appliedto the column, maximizing the linear flow velocity and compressing thegradient time to produce a peak production of at least 1 peak/2 seconds.Prior to this invention, it was believed that manipulating theseparameters beyond the levels described in the prior art wouldsignificantly decrease the resolution of the peaks eluting off thecolumn to an extent that it would not be possible to obtain discreteseparation of a mixture of small organic compounds. For instance,combinatorial libraries containing small organic molecules are oftenseparated with gradient reverse phase HPLC using a run time ofapproximately five to twenty minutes for quality control analysis. Itwas believed, according to the prior art, that reducing the total runtime by decreasing column volume and increasing linear flow velocitywould produce an equivalent loss in resolution. Surprisingly it wasdiscovered according to the invention that run time could be reduced byfive fold over the minimum run time which has been described in theprior art with only a minimum loss in resolution, based on peakcapacity, if the gradient time is also decreased. This finding indicatesthat the methods of the invention can produce much faster separationswith minimum reduction of peak capacity than those seen in the priorart. The parameter of peak capacity is defined as the number of baselineseparated peaks that will fit within the time that the gradient ischanged from a low percent of solvent B to a high percent of solvent B.In order to normalize between different analyses, the peak capacitydivided by the gradient time is defined as the “peak production”, andexpressed in units of peaks/second. Using the methods of the inventionat least 1 peak/2 seconds can be resolved. Preferably 1 peak/1 secondand even more preferably 1 peak/0.5 seconds are resolved using themethods of the invention. Resolution is the ability to distinguishindividual compounds eluting from the column. Adequate resolutionaccording to the invention is the ability to resolve 1 peak every 2seconds. As used herein, this means that the peak width at baseline ison average not more than 2 seconds. A determination of resolution usinga measure of baseline peak width is found in L. R. Snyder, J. J.Kirkland, J. L. Glajch, “Practical HPLC Method Development” 2^(nd)Edition, John Wiley & Sons, Inc., (1997).

As will be apparent from FIG. 1 and the description above, severalvariables will affect the peak production capacity of the method.Amongst these are (1) size of the column, (2) the packing material usedin the column, (3) the use of a full gradient in a minimum volume, (4)the total volume of liquid passing over the column, and (5) the linearflow velocity. These variables should be adjusted, as further describedbelow, to produce a rapid method having a maximum run time of one minuteand a peak production of at least 1 peak/2 seconds.

In preferred embodiments, the column used according to the method of theinvention is short and wide. Preferably the column has a length of lessthan 30 mm. A shorter column allows for a higher flow rate. With longercolumns, flow rate must be reduced in order to minimize the backpressure which is created within the column. Additionally a wide column,such as a column having an internal diameter of greater than 4 mm, ispreferred in order to minimize extra column band broadening associatedwith other parts of the instrumentation. However, a column having anywidth may be used according to the methods of the invention. A preferredwidth for analytical HPLC is between 4 and 5 mm. A preferred width forpreparative isolation is between 20 and 30 mm.

The packing material used in the column is a solid support particle withreverse phase properties. Preferably, the packing material has aparticle size of less than 5 μm and more preferably less than 4 μm. Suchpacking materials are commercially available and are well known to thoseof skill in the art. It is possible that improved packing materials willbe developed and in such case the preferred particle size may varydepending on the improvement in the materials.

The appropriate volume of solvent applied to the column is an importantparameter to the method of the invention. Ordinarily, approximately 30column volumes of solvent is applied to a column in a full gradient HPLCanalysis and often more for preparative isolation. The maximum volumeused according to the invention is 15 column volumes and preferably 8column volumes to maximize speed. A graph depicting the maximum volumeof liquid applied according to the methods of the invention in units ofcolumn volume is presented in FIG. 2. The volumes used in prior artmethods is presented in parentheses below the volume of the invention.As shown in the figure a complete gradient is applied to the columnwithin 10 column volumes over a time course of 20-40 seconds. Prior artgradients require 15-30 column volumes to achieve a complete gradient. Acleaning cycle of 2 column volumes of 95% acetonitrile or other elutionsolvent is used to flush any remaining molecules from the column over atime period of approximately 5-10 seconds. The column is thenequilibrated within 1 column volume and subjected to initial solventconditions for one column volume over a total time period ofapproximately 5-15 seconds.

The preferred mixing volume of the solvent is the minimum volume thatpermits a homogenous mixture of solvent A and solvent B. The minimumvolume is achieved by utilizing a small volume mixing chamber andminimum volume of tubing. An appropriate mixing chamber for theanalytical embodiment has an internal volume of less than 250 μl andpreferably less than 50 μl. Although such small mixing chambers are notcommercially available, 50 μl post-column reaction chambers, which arecommercially available can be used as a mixing chamber. In addition toselecting a mixing chamber having a minimum size, it is preferred thatthe mixing chamber be static. A static mixing chamber as used herein isa chamber or column packed with beads, usually made of steel or glass.As the solvent moves over the beads, it is subjected to turbulence andcaused to be mixed together.

It is important that the method of the invention be performed using acomplete gradient in order to assure that all components injected on thecolumn have been removed from the column prior to the injection of thenext sample. A “complete gradient” as used herein is a gradient ofsolvent which begins with a low percentage of solvent B (solvent B is anon-polar solvent such as acetonitrile) and a high percentage of solventA (solvent A is the aqueous phase). A low percentage of solvent B ispreferably below 20%. The percentage of solvent A and B is shifted withtime to produce a high percentage of solvent B and a low percentage ofsolvent A. A high percentage of solvent B is preferably above 70%.

The flow rate and the mixing volume dictate the time for the twosolvents to reach the HPLC column, which in our analytical system ismuch less than 0.1 minutes. Using a 50 μl mixing chamber, minimaltubing, and a flow rate of 3-5 ml/min the movement of the solvent fromthe pumps to the column would be complete in 2-6 seconds. Anotherimportant parameter is the linear flow velocity, the velocity with whichthe solvent moves through the column. The linear flow velocity isdependent on the flow rate and the internal diameter of the column.Preferably the linear velocity is greater than 3 mm/sec.

An additional parameter that can be used to increase peak capacity istemperature. As demonstrated in the examples below, when the temperatureof the sample through the continuous liquid path is increased over 20°C., the peak capacity is significantly increased. When combined with thehigh flow rates of the invention, the methods performed under hightemperatures provide significant increases in peak capacity. Thus,although the methods of the invention may be performed at anytemperature suitable for HPLC analysis, higher temperatures may bepreferred to increase the peak capacity of the system. In someembodiments, the temperature may be a temperature greater than or equalto 20° C., 30° C., 40° C., 50° C. or 60° C. When selecting anappropriate temperature for the HPLC method, one of ordinary skill inthe art will be aware that some compounds may be unstable at highertemperatures. Those of ordinary skill in the art will be able toidentify those compounds which are unstable at high temperatures.

The method of the invention is useful for separating small organiccompound mixtures. The mixture of compounds can include a number ofsmall organic compounds of unknown composition having variations inhydrophobicity. The method of the invention has the capability toresolve at least 30 compounds that exhibit different hydrophobicitycharacteristics, as identified by the gradient composition at which eachcompound elutes, in one minute or less. Under conditions in which theresolution power of the method results in a peak production of 1 peak/1second, the method of the invention has the ability to resolveapproximately 60 such compounds in one minute.

Usually, the mixture of compounds includes much less than the maximumnumber of compounds capable of being resolved by the system. Forinstance, a preferred mixture of compounds includes reactants and asubstantially pure product of the reactants. A mixture of compoundscontaining a “substantially pure product of the reactants” as usedherein is a mixture containing primarily the intended product, a smallamount of unreacted starting materials, as well as a few (preferablyless than 5) side products in significantly lower quantity than theintended product. Such a mixture is achieved by using reactants whichare only capable of producing a limited number of products under thegiven reaction conditions. In a preferred embodiment of the invention,the mixture of compounds is one derived from the preparation of acombinatorial library. A “combinatorial library of small organiccompounds” is a collection of closely related analogs that differ fromeach other in one or more points of diversity and are synthesized byorganic techniques using multi-step processes. Combinatorial librariesinclude a vast number of small organic compounds, some of which may haveimportant biological activity.

One type of combinatorial library, which is preferred according to theinvention, is prepared by means of parallel synthesis methods to producea compound array. A “compound array” as used herein is a collection ofcompounds identifiable by their spatial addresses in Cartesiancoordinates and arranged such that each compound has a common molecularcore and one or more variable structural diversity elements. Thecompounds in such a compound array are produced in parallel in separatereaction vessels, with each compound identified and tracked by itsspatial address. Regardless of the relative amount of starting materialsand products that are in each reaction vessel after reaction, the methodof the invention can be used to analyze the extent of reaction and/orisolate the components. Examples of parallel synthesis mixtures andparallel synthesis methods are provided in U.S. Ser. No. 08/177,497filed Jan. 5, 1994 and its corresponding PCT published patentapplication W095/18972, published Jul. 13, 1995 and U.S. Pat. No.5,712,171 granted Jan. 27, 1998 and its corresponding PCT publishedpatent application W096/22529.

Once a series of small organic compound mixtures are developed, themixtures can be separated and analyzed to determine the product formedand the extent of reaction. Each mixture of compounds represents aseparate sample that is injected onto an HPLC column. As shown in FIG. 1each sample is held within a well of a microtitre plate 24 in anautosampler 22. The samples are loaded and injected manually orautomatically using equipment controlled by a computer 32. The automaticloading and injection of the samples is preferred because it enables thecontinuous loading of samples at a rate which will not limit the overallprocess of analysis.

Prior to loading the sample on the column the column is conditioned byflowing through it the intended mobile phase. As discussed above, thecolumn is packed with a non-polar stationary phase on solid supportparticles, and can be obtained from a variety of commercial sources.HPLC columns can be obtained from a variety of commercial sources suchas MACMOD (Chaddsford, Pa.). Once the column is conditioned, the systemis initiated by the injection of the sample. As the small organiccompounds contact the non-polar packing material each molecule isadsorbed to the packing material. The affinity with which each compoundadsorbs to the packing material is dependent on the hydrophobicity ofthe individual compound. The injection of the sample defines time zerofor the run.

A gradient is applied to the column immediately after the injection ofthe sample in order to elute the compounds bound therein in distinctfractions. A gradient HPLC system includes two reservoirs, 12 and 14,each containing a different polarity solvent which are pumped through amixing chamber 16 and over the column 20 by means of a pump. In apreferred embodiment, solvent flow is maintained by a non-pulsating HPLCpump such as that available as part of a Shimadzu (Columbia, Md.) HPLCsystem. A full gradient from 15% to 95% of acetonitrile is then appliedto the column. The compounds in the mixture injected on the column whichare polar have a greater affinity for the initial composition of theeluant than the stationary phase and are thus eluted more rapidly thanthe more non-polar compounds which have a greater affinity for thehydrophobic stationary phase. Solvents typically used for gradients inreversed phase HPLC generally include acetonitrile, methanol,isopropanol and propanol. Modifiers are typically added to the mobilephase, primarily to buffer the pH to a certain narrow range, and includea variety of acids and bases such as phosphoric acid, perfluorinatedcarboxylic acids and amines.

An HPLC compatible detector is used to detect the presence of smallorganic compounds as they are eluted from the column. A compatibledetector is one which is capable of detecting a signal from a compoundin an eluant and which produces a signal to indicate the presence ofthat compound. The detector should allow data acquisition at a rate ofgreater than 10 points per second and preferably greater than 20 pointsper second. HPLC compatible detectors include, but are not limited to,fluorescent, electrochemical, IR, NMR, chemiluminescent, UV and massspectrometry. Preferably the HPLC compatible detector is a UV detector28, since commercially available UV detectors are capable of achievingthe required data acquisition rate when the settings are adjusted toachieve maximal values. Furthermore, UV detectors are generallyapplicable to a large and diverse number of chemical clauses and a largevariety of mobile phases.

In some cases, the HPLC compatible detector is both a UV detector 28 anda mass spectrometer 30. The use of both a UV detector and a massspectrometer is preferred because it allows the methods of the inventionto achieve, both purity (by UV) and structural (by MS) information foreach separated and detected component being eluted from the column.Moreover, the mass spectrometer can be used as a high specificitymulti-dimensional detector to provide general information on a class ofcompounds or specific information on a particular compound. Thesebeneficial properties coupled with the inherent high sensitivity of themass spectrometer make it one of the most desirable detectors to becoupled with the separation power of HPLC.

In the aspect of the invention described above the method may be usedfor analysis, such as quality control analysis and/or for preparativeisolation of at least one component of a mixture of compounds. In thisaspect of the invention one or more compounds may be collected afterseparation on the column in distinct fractions. The HPLC compatibledetector is used to identify the presence of a compound in each fractionand the fractions are separated into an acceptable container such as atube or a well of a microtitre plate.

In another aspect the invention is a rapid high performance liquidchromatography method for the preparative isolation of a concentratedfraction of a small organic compound from a mixture of compounds. Eachof the parameters described above in relation to the method ofseparation are also applicable to this aspect of the invention. Themethod in this aspect of the invention differs, however, from the abovemethod in that the separation of molecules is performed only for thepurpose of separating compounds in a mixture into distinct fractions andthe compounds are collected for future analysis or use. The method isperformed as described above except that the elution permits resolutionwith a peak production of at least 1 peak/4 seconds. In some embodimentsthe elution permits resolution with a peak production of at least 1peak/2 seconds.

Preferably the compounds are collected in fractions having a volume of 2milliliters or less. The methods of the invention accomplish theseparation of compounds in such a short time that the total volume thateach compound elutes in 2 milliliters or less. This is advantageousbecause the compound of interest is present in a fairly concentratedform as opposed to the prior art methods where the compound of interestis often eluted in a minimum volume of 4 milliliters.

As described above, many variations on these particular examples arepossible and, therefore, the examples are merely illustrative and notlimiting of the present invention.

EXAMPLES Example 1 Gradient Reverse Phase HPLC Analysis of StandardMixtures of Small Organic Molecules

Equipment: All analytical separations were performed on a Shimadzu(Columbia, Md.) HPLC System consisting of two LC-10AS pumps, a SIL-10Aautosampler, a SCL-10A system controller and a SPD-10A UV-detector. Thesystem was modified for high performance application in the followingmanner. A low volume static mixer from Supelco (Bellefonte, Pa.) with a250 ul cartridge was used for fast and efficient high pressure mixing ofthe eluents. In combination with the short 30 mm columns a 50 ul mixingcartridge was used to further minimize gradient delay. The originalbypass of the Shimadzu autoinjector was removed to eliminate bandbroadening effects. Injection was performed in the partially filled loopmode, injecting between 1 and 5 ul in a 50 ul loop. Connections betweeninjector, column and detector were made with 0.007″ tubing and thelength kept at a minimum to prevent extra column band broadening. Theoriginal flow-cell of the UV detector was replaced with a semi-microversion, having a path length of 5 mm and a smaller total volume of 2.5ul. The detector response was set to 1 to allow for detection of rapidlyeluting, narrow peaks. Data acquisition was performed by theChromperfect software from Justice Innovations (Palo Alto, Calif.) at arate of 20 points/second.

Chemicals: Water and acetonitrile were HPLC grade, dimethylsulfoxide wasA.C.S. grade from Baker (Philipsburg, N.J.). All other chemicals werethe highest available purity grade from Aldrich (Milwaukee, Wis.). TheHPLC eluents were prepared by adding 0.1% (v/v) trifluoroacetic acid towater (solvent A) and acetonitrile (solvent B).

HPLC Columns: The Zorbax SB-C8 columns (50 mm×4.6 mm and 30 mm×4.6 mmboth packed with 3.5 μm particles; 150 mm×4.6 mm packed with 5 μmparticles) were obtained from MACMOD (Chaddsford, Pa.). The ProntosilC18-SH columns (50 mm×4.6 mm, 3 μm particles) was a gift from BischoffAnalysentechnik (Leonberg, Germany).

An example of a large difference in compound hydrophobicity isrepresented by our standard test mixture, which consists of thefollowing components: acetamidophenol, 2-hydroxydibenzofuran andt-butylphenoxybenzaldehyde. In a reverse phase HPLC separation, theacetamidophenol elutes during the initial part of the gradient, the2-hydroxydibenzofuran elutes in the middle of the gradient and thet-butylphenoxybenzaldehyde elutes at the end of the gradient. A mixtureof the three compounds was prepared in dimethylsulfoxide (DMSO). Themixture was then subjected to HPLC analysis according to the methods ofthe invention under the condition described above:

The test mixture was first analyzed with a column packed with reversephase silica having 3.5 μm particle size, at a flow rate of 3 ml/min inaddition to each of the above conditions. The compounds were eluted witha gradient of 15-95% acetonitrile applied to the column in 0.7 min with10 second hold and 5 second equilibration time. The results are shown inFIG. 3. Three major peaks representing each of the three components ofthe test mixture were resolved in less than 1 minute. Based on thebaseline peak width of the middle peak, approximately 60 compounds withvarying hydrophobicities could be baseline resolved with this method.

The same mixture of compounds was analyzed with a column packed withreverse phase silica having 3.5 μm particle size, at a flow rate of 5ml/min in addition to each of the above conditions. The molecule were,again, eluted with a gradient of 15-95% acetonitrile applied to thecolumn in 0.3 min with a 10 second hold and 5 second equilibration time.The results are shown in FIG. 4. Once again based on the width of themiddle peak, approximately 30 compounds of varying hydrophobicitiescould be baseline resolved with this 30 second method.

Example 2 Gradient Reverse Phase HPLC Analysis of Combinatorial LibrarySamples Obtained by Parallel Synthesis

One sample from two different combinatorial libraries prepared byparallel synthesis by methods such as those disclosed in U.S. Ser. No.08/177,497, filed Jan. 5, 1994 and its corresponding PCT publishedpatent application W095/18972, published Jul. 13, 1995 and U.S. Pat. No.5,712,171 granted Jan. 27, 1998 and its corresponding PCT publishedpatent application WO96/22529 were chosen for analysis using the methodsdescribed in Example 1. The results for sample A1990702D9 analyzed usinga traditional 20 minute analysis are compared to those obtained from the1 minute and 30 second analysis in FIG. 5. The 20 minute method used aZorbax SB-C8 (150×4.6 mm) packed with 5 μm particles and a full gradient(15%-95% solvent B) at 15 ml/min. Although the peak capacity is less forthe shorter analyses, the resolution is more than adequate to determinepurity, even in such a complex sample which would be the worst casescenario. Similarly, the results for sample AQ130QC48H5 using the 20minute, 1 minute and 30 second analyses are given in FIG. 6. Once again,the 1 minute method provides the same purity information as the longer20 minute analysis. Moreover, the 30 second analysis provides similarinformation. Although the small peak between the two major ones ispartially merged with the major component, there is still adequateresolution to indicate the presence of the minor component. Theforegoing written specification is considered to be sufficient to enableone skilled in the art to practice the invention. The present inventionis not to be limited in scope by the disclosed embodiments, since theseembodiments are intended merely as illustrative of particularembodiments of the invention as enabled herein and any methods that arefunctionally equivalent are within the scope of the invention. Indeed,various modifications of the invention in addition to those shown anddescribed herein will become apparent to those skilled in the art fromthe foregoing description and fall within the scope of the appendedclaims.

Example 3 Effect of Flow Rate on the Rapid Gradient Reverse Phase HPLCAnalysis of the Invention

In order to determine the effect of flow rate on the rapid analysismethods of the invention, the method was performed using various flowrates ranging from 2 to 4 ml/min. The data is shown in FIG. 7. In thecase of the very steep 1 minute gradient, an increase in the flow ratefrom 2 ml/min (resulting in a gradient volume of 2.4 column volumes) to4 ml/min (4.8 column volume) increases the peak capacity from 29 to 48.The larger gradient volume achieved by increasing the flow rate alsolowers the total analysis time as indicated by the elution of the lastpeak. The peak production rate in this case almost doubles with the flowrate from 0.47 to 0.8 peak/sec. The linear flow rate at 4 ml/min was 6.4mm/second and the back pressure was about 250 bar which is acceptablefor a routine application. Thus, columns can be run at very high linearflow rates with steep gradients in order to obtain a high peak capacityand peak production rate.

Example 4 Gradient Reverse Phase Preparative HPLC Analysis of StandardMixture of Small Organic Molecules

Equipment: Preparative separations were performed with a system of twoRainin Dynamax SD-1 pumps (Woburn, Mass.), a Gilson-215 (Madison, Wis.)configured as both an autosampler and fraction collector, a Shimadzu(Columbia, Md.) SPD-10A UV detector with a preparative cell. Injectionwas performed in the partially filled loop mode injecting 250 ul in a 5ml loop. Detection was at 254 nm and the detector response was set to 1to allow for detection of rapidly eluting, narrow peaks. Dataacquisition was performed by Unipoint software from Gilson (Madison,Wis.).

Chemicals: Water and acetonitrile were HPLC grade, dimethylsulfoxide wasA.C.S. grade from Baker (Philipsburg, N.J.). All other chemicals werethe highest available purity grade from Aldrich (Milwaukee, Wis.). TheHPLC eluents were prepared by adding 0.1% (v/v) trifluoroacetic acid towater (solvent A) and acetonitrile (solvent B).

HPLC Columns: YMC ODS-A C18, 20 mm×50 mm packed with 5 um particlesobtained from YMC (Wilmington, N.C.).

The standard test mixture of acetamidophenol, 2-hydroxydibenzofuran, andt-butylphenoxybenzaldehyde was prepared at 40 mg/ml of each component inDMSO. The mixture was then subjected to preparative HPLC analysis. Theflow rate of the mobile phase was 80 ml/min and the compounds wereeluted with a gradient of 10%-95% acetonitrile applied to the column in1 minute with a 20 second hold and a 40 second equilibration time. Thefraction collector was set to collect the second and third peak. Theresults are shown in FIG. 8. Based on the baseline peak width,approximately 30 compounds with varying hydrophobicities could beresolved and collected with this method.

Example 5 Gradient Reverse Phase Preparative HPLC Analysis of aCombinatorial Library Sample

One sample from a combinatorial library prepared by parallel synthesisby methods such as those disclosed in U.S. Ser. No. 08/177,497, filedJan. 5, 1994 and its corresponding PCT published patent applicationW095/18972, published Jul. 13, 1995 and U.S. Pat. No. 5,712,171 grantedJan. 27, 1998 and its corresponding PCT published patent applicationWO96/22529 were chosen for analysis using the method described inExample 4. However, the detection was at 217 nm and 400 ul of a 30 mMsolution of the sample was injected. The fraction collector was set tocollect the expected product peak, which was identified as the last peakin the chromatogram. The results of sample A1990703D9 are shown in FIG.9.

Example 6 Effects of Temperature on Rapid Gradient Reverse Phase HPLCAnalysis

In order to determine the effect of temperature on the rapid analysismethods of the invention, the method was performed using varioustemperatures ranging from 20° C. to 60° C. under conditions such asdifferent flow rates. The data is presented in Table I. As shown in thetable, the peak capacity increases significantly when the temperature israised from 20 to 60° C. and this increase in peak capacity is even morepronounced when the system is run at high flow rates. As shown in thetable, the peak capacity of a sample run on a 30 mm column at 4 ml/minusing a 1 minute method with a 0.75 minute gradient, increased from 44at 25° C. to 55 at 60° C. This correlates to a 25% increased based on acorresponding decrease in peak width. In a similar experiment in whichthe flow rate and temperature are increased from 4 ml/minute to 5ml/minute and from 25° C. to 60° C. respectively, the peak capacityincreases from 44 to 60, which is a 35% increase. In addition to thechanges in peak capacity, the eluent viscosity is decreased at highertemperatures. Faster flow rates at comparable pressure drops may also beaccomplished at higher temperatures. For instance, a decrease inpressure of 50% was observed when the temperature was raised from 20 to60° C. Additionally, a 5-10% increase in velocity of eluent wasobserved, indicated by a decrease in retention time of the compound.

TABLE 2 4.6 × 30 mm column 4 ml/min 5 ml/min 4 ml/min 6 ml/minpeak-capacity 25 C. 25 C. 60 C. 60 C. 1.00 minute method 44 47 55 600.75 min gradient 0.75 minute method 37 38 44 48 0.50 min gradient 0.50minute method 29 30 34 39 0.35 min gradient 4.6 × 50 mm column 4 ml/min4 ml/min 6 ml/min peak-capacity 20 C. 60 C. 60 C. 1.00 minute method 3753 59 0.75 min gradient 0.75 minute method 46 0.50 min gradient

During the experimentation concerning the effects of temperature, it wasdiscovered that the existing equipment could not effectively carry outthe appropriate temperature change without some modification. Initiallya column heater jacket was used in order to regulate the temperature.The column is immersed in a circulating liquid which is temperaturecontrolled. The eluents were brought to the desired temperature throughheat exchange capillaries that were immersed in a system regulated by athermostat. It was found, however, that the temperature of the eluentdropped from approximately 50° C. to about 28° C. between the mixingdevice and the column at the high flow rates required for the rapid HPLCanalysis of the invention. A modified version of the equipment wasarranged by immersing a manual injector valve together with the heatexchanger and the column in a water bath of the desired temperature forthe experimental condition. This modification to the equipment provideda homogenous temperature distribution throughout the liquid path andenabled the characterization of temperature effects described above.Those of ordinary skill in the art will be able to modify the existingequipment to accomplish these effects based on this teaching.

We claim:
 1. An improved rapid method for the separation of smallorganic compounds in a mixture of compounds by applying the mixture ofcompounds to a reverse phase column (20) in a gradient high performanceliquid chromatography system, applying a complete gradient to the column(20) in a maximum volume; and causing each small organic compound withinthe mixture of compounds to elute in a distinct fraction from the column(20), and wherein the maximum volume is 10×column volume, and elutingeach small organic compound such that the elution permits resolutionwith a peak production of at least 1 peak/2 seconds.
 2. The method ofclaim 1, further comprising the step of detecting at least one of thecompounds as it elutes from the column (20).
 3. The method of claim 2,wherein the method is a method for analysis of at least one compound inthe mixture of compounds.
 4. The method of claim 1, further comprisingthe step of collecting at least one of the compounds in a distinctfraction as it elutes from the column (20).
 5. The method of claim 4,wherein the method is a method for preparative isolation of at least onecompound in the mixture of compounds.
 6. The method of claim 4, whereinthe at least one compound is collected in a fraction having a volume of2 milliliters or less.
 7. The method of claim 1, wherein the mixture ofmolecules includes reactants and a substantially pure product of thereactants.
 8. The method of claim 1, wherein the column (20) is lessthan or equal to 30 mm in length.
 9. The method of claim 1, wherein thecolumn (20) has a packing material which has an average diameter of lessthan 5 microns.
 10. The method of claim 1, wherein the peak productionis at least 1 peak/1 second.
 11. The method of claim 1, wherein the peakproduction is at least 1 peak/0.5 seconds.
 12. The method of claim 1,wherein a total volume of liquid applied to the column is less than15×column (20) volumes.
 13. The method of claim 1, further comprising acleaning volume having a maximum of 2×column volume.
 14. The method ofclaim 1, further comprising an equilibration volume having a maximum of1×column volume.
 15. The method of claim 1, wherein the mixture ofcompounds is applied to the column (20) at a first time point andwherein all compounds are eluted within a time period of less than oneminute from the first time point.
 16. The method of claim 15, whereinall compounds are eluted within a time period of less than 30 seconds.17. The method of claim 15, wherein all compounds are eluted within atime period of less than 20 seconds.
 18. The method of claim 1, whereinthe mixture of molecules is a member of a combinatorial library of smallorganic molecules.
 19. The method of claim 18, wherein the combinatoriallibrary is made by means of parallel synthesis methods.
 20. The methodof claim 1, wherein the column has a linear velocity of at least 3mm/sec.
 21. The method of claim 1, wherein the method is a method foranalysis of at least one compound in the mixture of compounds.
 22. Themethod of claim 1, wherein the method is a method for preparativeisolation of at least one compound in the mixture of compounds.
 23. Animproved rapid high performance liquid chromatography method for thepreparative isolation of a concentrated fraction of a small organiccompound from a mixture of compounds by applying the mixture ofcompounds to a reverse phase column (20) in a gradient high performanceliquid chromatography system, applying a complete gradient to the column(20) in a maximum volume; and causing the small organic compound toelute in a distinct fraction, separate from the other molecules, fromthe column (20), and collecting the small organic compound, and, whereinthe maximum volume is 10×column volume, and eluting the small organiccompound such that the elution permits resolution with a peak productionof at least 1 peak/4 seconds.
 24. The method of claim 23, wherein themixture of molecules includes reactants and a substantially pure productof the reactants.
 25. The method of claim 23, wherein the column (20) is20-30 mm in length.
 26. The method of claim 23, wherein the column (20)has a packing material which has an average diameter of less than 5microns.
 27. The method of claim 23, wherein a total volume of liquidapplied to the column (20) is less than 15×column volumes.
 28. Themethod of claim 23, further comprising a cleaning volume having amaximum of 2×column volume.
 29. The method of claim 23, furthercomprising an equilibration volume having a maximum of 1×column volume.30. The method of claim 23, wherein all compounds are eluted within atime period of less than 60 seconds.
 31. The method of claim 23, whereinthe mixture of molecules is a member of a combinatorial library of smallorganic molecules.
 32. The method of claim 31, wherein the combinatoriallibrary is made by means of parallel synthesis methods.
 33. The methodof claim 23, wherein the column (20) has a linear velocity of at least 3mm/sec.
 34. The method of claim 23, wherein the small organic compoundis collected in a fraction having a maximum volume of 2 milliliters.